Bottom Line:
Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development.Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain.This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development.

fig6: Failure to Target the PCGF1/PRC1 Complex Leads to a Loss of H2AK119ub1, PRC2, and H3K27me3(A) Snapshots of ChIP-seq traces for KDM2B, RING1B, and SUZ12 in the Kdm2bfl/fl cells prior to (−OHT) and after 72 hr (+OHT) of tamoxifen treatment at the Epha7 and Klrg2 genes.(B) Heat map analysis of RING1B peaks (n = 3,488), showing ChIP-seq data for KDM2B, RING1B, and SUZ12 covering a 10 kb region centered over the RING1B peak −OHT and after 72 hr +OHT.(C) Log2 fold changes in normalized read counts comparing the ChIP-seq and RNA-seq signal −OHT and after 72 hr +OHT.(D) A Venn diagram showing all RING1B peaks (light blue), intersected with RING1B or SUZ12 peaks that have a greater than 1.5-fold reduction in RING1B/SUZ12 occupancy after 72 hr +OHT treatment (ΔRING1B [dark blue] and ΔSUZ12 [green]).(E) A box and whisker plot indicating the log2 fold change in RING1B and SUZ12 occupancy at sites that have RING1B changes of greater than 1.5-fold (ΔRING1B) or less than 1.5-fold (No Change) following 72 hr +OHT treatment. The significance of the changes in SUZ12 occupancy at these sites is indicated above the plot.(F) A scatter plot comparing the log2 fold change of RING1B and SUZ12 at RING1B sites in the Kdm2bfl/fl cells −OHT and after 72 hr +OHT.(G) A box and whisker plot indicating log2 fold change in gene expression at sites described in (E).(H) A scatter plot comparing the log2 fold change of gene expression to the fold change in RING1B occupancy at sites that show RING1B alterations.(I) ChIP analysis for polycomb factors and modifications at regions showing loss of RING1B, no significant loss of RING1B, and a nontarget site (NTS). All ChIP experiments were performed in biological triplicate with error bars showing SEM.(J) Gene expression analysis for the target genes analyzed by ChIP in (I). RT-PCR was performed in biological triplicate. Error bars show SEM.

Mentions:
To identify polycomb sites that are dependent on KDM2B-mediated targeting for RING1B binding, RING1B ChIP-seq was carried out in the Kdm2bfl/fl and tamoxifen treated cells. Removal of the KDM2B ZF-CxxC domain resulted in a widespread reduction of RING1B chromatin binding (Figures 6A–6D). Of the 3,488 high-confidence RING1B peaks identified in ESCs, 43% showed a greater than 1.5-fold reduction in RING1B occupancy after tamoxifen treatment (Figure 6D), suggesting that a subset of RING1B-bound CpG islands is most sensitive to KDM2B loss, and other PRC1 complexes must contribute to RING1B occupancy at the remaining sites. Importantly, sites exhibiting RING1B loss also showed reduced H2AK119ub1 levels, consistent with a role for PCGF1/PRC1 in catalyzing this modification (Figure 6I).

fig6: Failure to Target the PCGF1/PRC1 Complex Leads to a Loss of H2AK119ub1, PRC2, and H3K27me3(A) Snapshots of ChIP-seq traces for KDM2B, RING1B, and SUZ12 in the Kdm2bfl/fl cells prior to (−OHT) and after 72 hr (+OHT) of tamoxifen treatment at the Epha7 and Klrg2 genes.(B) Heat map analysis of RING1B peaks (n = 3,488), showing ChIP-seq data for KDM2B, RING1B, and SUZ12 covering a 10 kb region centered over the RING1B peak −OHT and after 72 hr +OHT.(C) Log2 fold changes in normalized read counts comparing the ChIP-seq and RNA-seq signal −OHT and after 72 hr +OHT.(D) A Venn diagram showing all RING1B peaks (light blue), intersected with RING1B or SUZ12 peaks that have a greater than 1.5-fold reduction in RING1B/SUZ12 occupancy after 72 hr +OHT treatment (ΔRING1B [dark blue] and ΔSUZ12 [green]).(E) A box and whisker plot indicating the log2 fold change in RING1B and SUZ12 occupancy at sites that have RING1B changes of greater than 1.5-fold (ΔRING1B) or less than 1.5-fold (No Change) following 72 hr +OHT treatment. The significance of the changes in SUZ12 occupancy at these sites is indicated above the plot.(F) A scatter plot comparing the log2 fold change of RING1B and SUZ12 at RING1B sites in the Kdm2bfl/fl cells −OHT and after 72 hr +OHT.(G) A box and whisker plot indicating log2 fold change in gene expression at sites described in (E).(H) A scatter plot comparing the log2 fold change of gene expression to the fold change in RING1B occupancy at sites that show RING1B alterations.(I) ChIP analysis for polycomb factors and modifications at regions showing loss of RING1B, no significant loss of RING1B, and a nontarget site (NTS). All ChIP experiments were performed in biological triplicate with error bars showing SEM.(J) Gene expression analysis for the target genes analyzed by ChIP in (I). RT-PCR was performed in biological triplicate. Error bars show SEM.

Mentions:
To identify polycomb sites that are dependent on KDM2B-mediated targeting for RING1B binding, RING1B ChIP-seq was carried out in the Kdm2bfl/fl and tamoxifen treated cells. Removal of the KDM2B ZF-CxxC domain resulted in a widespread reduction of RING1B chromatin binding (Figures 6A–6D). Of the 3,488 high-confidence RING1B peaks identified in ESCs, 43% showed a greater than 1.5-fold reduction in RING1B occupancy after tamoxifen treatment (Figure 6D), suggesting that a subset of RING1B-bound CpG islands is most sensitive to KDM2B loss, and other PRC1 complexes must contribute to RING1B occupancy at the remaining sites. Importantly, sites exhibiting RING1B loss also showed reduced H2AK119ub1 levels, consistent with a role for PCGF1/PRC1 in catalyzing this modification (Figure 6I).

Bottom Line:
Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development.Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain.This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development.